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Dear All,
I am working on sustained-release formulation of insulin using PLGA
microspheres. Phosphate buffer pH 7.4 (0.05 M) is used as release
medium and
insulin is determined by HPLC. The cumulative concentration of insulin
decreases significantly after 48 hours and in some samples after 24
hour. I
guess insulin tends to be aggregated in 37oC release medium and
aggregated
insulin is trapped in precolumn of HPLC. I am wondering whether one
of you
has the same experience. Any helpful suggestion for overcoming the
problem
would be much appreciated.
Regards,
Hamed
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Dear Hamed,
I was reading up on similar issues a while ago. I was looking for
solutions to the problem of aggregation of monomeric insulin in
buffers at physiological pH and temperature. I found out through
reading that this problem is not uncommon. The "formulation"
solutions proposed are still under patent. The patents are registered
in the US under the following numbers: 6734162 and 6,551,992.
Summaries can be accessed at: http://www.pharmcast.com/Patents/Yr2003/
April2003/042203/6551992_Insulin042203.htm
I hope this is of help.
Murad Melhem, Ph.D.
Cognigen Corporation
Buffalo, NY
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You can probably disrupt using ionic and non ionic detergents or, since
the xmerization/aggregation is salt (Zn and Ca dependent a chelating
agent may do)
http://www.ysbl.york.ac.uk/~mgwt/thesis-tth/chapter5.html
Ed O'Connor, PhD
Technical Director, Immunoanalytical
Tandem Laboratories
115 Silvia Street
West Trenton, New Jersey
609-228-0243
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Insulin tends to stick to the tube walls, it will be preferable to
perform these experiment with BSA or serum in the release medium.
Moreover human insulin can be easily quantitate by ELISA.
Dr Roberto Conti
Sigma-Tau
Dep. of Endocrinology and Metabolism
00040 pomezia, Roma
Phone: +39-06-91393322
Fax: +39-06-91393988
e-mail. roberto.conti.at.sigma-tau.it
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Dear Dr. Connor,
I included CTAB as cationic surfactant and tween 20 nonionic one 0.1% in
dissolution medium and also double distilled water (for HPLC)
(completely
free of salts) used for buffer preparation. But I met the problem again.
Sincerely yours,
Hamed
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Dear Dr. Conti,
I carried out the experiments with glass and also polypropylene tubes
(which
is recommended in articles as a suitable tube to prevent protein
adhesion to
tube walls) but there is no difference. Because of large molecular
weight of
BSA, it may be trapped by column (C18 10u bondapack 25 cm) therefore
I can
not use it. The determination range of ELISA is 0-10 ng/ml which is very
narrow for in-vitro assay of insulin. Lowry assay is suitable but color-
based determination methods are not accurate enough.
Sincerely yours,
Hamed
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